Potentially hazardous levels of heavy metals have dispersed into subsurface sediment and groundwater in a number of metal-contaminated sites and represent a challenge for environmental restoration. This contamination can cause severe risks to human health due to direct metal uptake into vegetables and due to spreading via wind or via infiltration in the groundwater or via flooding. The concentrations and distribution of metals are influenced by the intrinsic binding capacity of the soil via ion-exchange (on clay), adsorption to functional groups (on organic matter), precipitation (due to pH changes) and co-precipitation (on iron oxy-hydroxides), as well as integration in the mineral lattice. In addition, micro-organisms play a role in the fate of metals resulting in such bio-geochemical processes as biosorption on biomass, bioprecipitation due to oxidation or reduction reactions, precipitation with sulfides, obtained through sulfate reduction etc.
Effective bioremediation of these sites requires knowledge of genetic pathways for resistance and biotransformation by component organisms within a microbial community. However, a comprehensive understanding of bacterial mechanisms of heavy metal toxicity and resistance has yet to be achieved. While many metals are essential to microbial function, heavy metals, i.e., most of those with a density above 5 g/cm3, have toxic effects on cellular metabolism.
The majority of heavy metals are transition elements with incompletely filled d orbitals providing heavy metal cations which can form complex compounds with redox activity. Therefore, it is important to the health of the organism that the intracellular concentrations of heavy metal ions are tightly controlled. However, due to their structural and valence similarities to nontoxic metals, heavy metals are often transported into the cytoplasm through constitutively expressed nonspecific transport systems. Once inside the cell, toxic effects of heavy metals include nonspecific intracellular complexation, with thiol groups being particularly vulnerable. Interactions of these nonspecific complexes with molecular oxygen leads to the formation of reactive oxygen species such as H2O2, resulting in oxidative stress within the cell. In addition to oxidative stress, complexation of sulfhydryl groups with heavy metal cations results in reduced activity of sensitive enzymes.
In order to achieve effective remediation, there is an acute need to develop new methods of assessing the heavy metal concentrations in natural and industrial environments. Existing methods for heavy metal detection including spectroscopical methods, such as AAS, AES, ICP-MS, etc.; or electrochemical methods, such as ISE, polarography, etc. These methods, however, can be expensive or not useful when there is a need to detect metals at low concentrations. Moreover, these methods can only detect the total amount of heavy metals and not the bioavailable concentrations accessible to the living organisms. Therefore, development of new and inexpensive methods for detection of bioavailable heavy metal concentrations is highly desirable.
Biosensors are useful analytical devices in this respect, and various have been described for heavy metal detection. Biorecognition elements may include whole cells, e.g. bacteria, fungi, lichens, mosses, etc.; or proteins, e.g. enzymes, apoenzymes, antibodies, etc. Transducers may be potentiometric, amperometric, optic, conductometric, spectrophotometric, etc. Currently available bioluminescent methods to assess ecotoxicity include naturally luminescent marine bacteria, primarily the MICROTOX system (AZUR Environmental).
An ideal assessment method for the presence of heavy metals is inexpensive, easy to use, and sensitive. Improved compositions and methods for assessment of toxic metals, including uranium, are of great interest. The present invention addresses these issues.
Relevant Literature
Liao et al. (2006) Environ Pollut. 142(1):17-23. Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors. Shetty et al. (2003) Anal Bioanal Chem. 376(1):11-7. Luminescence-based whole-cell-sensing systems for cadmium and lead using genetically engineered bacteria. Shetty et al. (2004) Biotechnol Bioeng. 5; 88(5):664-70. Fluorescence-based sensing system for copper using genetically engineered living yeast cells.
Hu et al. (2005) J Bacteriol. 187(24):8437-49. Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus. Southward and Surette (2002) Mol Microbiol. 45(5):1191-6. The dynamic microbe: green fluorescent protein brings bacteria to light.
U.S. Pat. No. 6,210,948, Expression and secretion of heterologous polypeptides from caulobacter. 